| Abstract|| |
Context: Clear cytology specimens are processed by cytocentrifugation which is preferred over membrane filters (MF). Although both techniques are expensive, cytocentrifugation is less tedious and may cause cellular distortion. Aim: To standardize “Agar cell-suspension” (ACS) an innovative, simple, cost-effective technique to process clear specimens. Methods and Materials: About 93 clear specimens (65 urine, 15 effusion, 12 CSF, and 1 bronchial lavage) were processed by both cytocentrifugation and ACS. The sample was centrifuged in two tubes; one was used for ACS and other for cytocentrifugation. ACS smears were prepared by mixing one drop of 0.5% agar solution with the last drop of the centrifugate. Smears were fixed in methanol and stained by Papanicolaou staining method. ACS smears were compared with cytocentrifuged smears (CS) and evaluated for cellularity, cytomorphology preservation, staining quality, time, and cost. Results: As compared to CS smears, ACS smears showed better cellularity in 16.1%, comparable in 53.7%, and less in 30.1%. All ACS smears (100%) showed well-preserved cytomorphology as compared to 96.7% CS. Staining quality was optimal in 96.7% ACS smears against 91.3% CS. Both techniques took equal time. The additional cost of ACS was only 0.03 INR compared to 12.50 INR for CS. Conclusions: ACS is an innovative, simple, easy, and cost-effective technique for processing clear specimens. It gives equally good results comparable to cytocentrifugation in terms of cellularity and staining quality. ACS does not cause cell distortion or air-drying as seen in some CS. Thus, ACS is a superior alternative to cytocentrifugation.
Keywords: Agar cell-suspension, clear samples, cytopreparatory techniques
|How to cite this article:|
Dighe SB, Ruben IC, Waghela CB. “Agar Cell-Suspension”: A novel technique for processing clear specimens. J Cytol 2020;37:26-9
| Introduction|| |
Cytology is a clinically oriented specialty that can flourish when a cytologist spends time and energy on the basic initial steps of planning good and meticulous smear preparation. A well-prepared smear is a backbone and the most basic step, without which even a knowledgeable and experienced cytologist is at loss and is not able to comment or give any opinion.
All fluid specimens (serous effusions, urine, CSF, and bronchial lavage) need to be centrifuged for cytological analysis. On centrifugation, these fluid specimens may either yield sediment or they may not yield any visible sediment. When sediment is produced, the smears are directly prepared from the sediment using a swab stick and subjected to further processing. If there is no visible sediment, then smears are generally prepared by concentrating the few cells by using sophisticated techniques such as cytocentrifugation or membrane filtration to increase the yield of the cells.,, Such fluid specimens, which do not produce any sediment on centrifugation, are also known as sparsely cellular fluids or clear fluids.
Both cytocentrifugation and membrane filtration are very expensive techniques and one needs to initially invest a huge amount in acquiring the equipment and also has to bear with the recurring cost of the filters in the membrane filtration and filter cards used in the cytocentrifugation process. Cytocentrifugation is preferred as it is less tedious and quick than the membrane filtration, but at times it may cause cellular distortion.
In India, membrane filtration and cytocentrifugation may not be available in all the center's especially in the rural setup or at the grassroot levels. So when such sophisticated equipment is not available for processing clear fluids, direct smears are made from the centrifugate. But there are some drawbacks in such preparation viz. lesser cells get transferred on the slide and the cells are dispersed in a larger area.
Thus, our aim was to standardize a manual, simple, cost-effective innovative technique, namely, ACS to process clear/sparsely cellular specimens and also to compare ACS smears along with cytocentrifuged specimens which were used as control smears.
| Materials and Methods|| |
For this new ACS technique, 0.5% of the Agar solution was used. 0.5 gm of agar powder was added to 100 mL of water, mixed well and then boiled to 100°C, till the solution becomes clear. It was allowed to cool at room temperature. The solution remains in a jelly-like or a semi-solid condition and is ready for use. The solution should be refrigerated and can be used for the next 15 days.
Around 93 consecutive clear specimens were prospectively selected for standardizing the new ACS technique. The specimens included (65 urine, 15 effusions, 12 CSF, and 1 bronchial lavage).
The specimen was thoroughly mixed and then taken in two centrifuge test tubes. Both the centrifuge tubes were then centrifuged at 2000 rpm for 10 min. One tube was subjected to the cytocentrifugation method and the other tube was subjected to the new ACS technique. For the cytocentrifugation method, the tube was decanted and only approximately 1 mL of the centrifugate was left behind in the tube. Two cytocentrifuged smears were then prepared by using this centrifugate.
For the new ACS method, the other tube was decanted completely and an excess amount of the supernatant was blotted [Figure 1]a so that only a single drop of the centrifugate was left behind. To this a single drop of 0.5%, Agar solution was added with the help of a Pasteur pipette [Figure 1]b. The solution was then mixed well and one drop of the solution was put on the slide [Figure 1]c and smears were prepared [Figure 1]d. Two such smears were prepared.
|Figure 1: Preparation of smear by ACS technique. (a) Blotting of excess fluid. (b) Add single drop of 0.5% agar solution to centrifugate. (c) Placeone drop of the mixture on the slide. (d) Prepare smears|
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Both the cytocentrifuged smears and ACS smears were fixed immediately in 100% methanol and both the smears were subjected to the routine Papanicolaou staining.
For Papanicolaou staining, the ideal fixative used is the ether-95% ethanol (1:1) solution. However, today various laboratories that cannot acquire tax-free alcohol have replaced this fixative of choice to other less expensive alternatives like 100% methanol or isopropanol. It has also been observed that the cell alterations produced by using these alternative fixatives are minimal.
Evaluation of smears
Two cytotechnologists evaluated smears prepared by these two methods individually.
Smears prepared from these two techniques viz. agar cell-suspension method, cytocentrifugation were assessed on the following criteria.
1. Cellularity: Cellularity of smears was assessed on the basis of the number of cells seen in the low power field and graded as 0 or acellular, +, ++, +++
+: 1-5 cells/lpf
++: 5-10 cells/lpf
The number of cells present in each ACS smear and the cytocentrifuged smear was counted and graded accordingly. Then the number of cases where the grades were same was counted and finally scored as “ACS has the same cellularity as cytocentrifuged smears (ACS = cytocentrifuged)” or “ACS had more cellularity than cytocentrifuge (ACS > cytocentrifuged)” or “ACS has less cellularity than cytocentrifuged (ACS < cytocentrifuged)”.
The smears also were evaluated to check the yield of the diagnostic cells.
2. Staining quality: Staining of cytoplasm and nucleus was considered for this criterion. Staining quality was assessed as good or optimum and poor or suboptimal.
The staining reaction was graded as good when nuclear chromatin, nuclear borders, nucleoli, cytoplasmic vacuoles, and cytoplasmic borders are visualized properly with optimum staining. Staining was graded as poor when staining is pale, faded, or deeply stained.
3. Preservation of smears: Smears showing well-preserved cell morphology were graded as good and those showing air-drying artifacts as poor.
4. Background staining: Smears were evaluated to check if the smears showed any background staining which could obstruct the cytomorphology of the diagnostic cells.
5. Time: Total time taken for smear preparation by each technique.
6. Cost: One-time cost for acquiring the equipment and recurring cost for the accessories were compared and calculated in rupees per sample.
| Results|| |
When the cellularity was compared, both ACS and cytocentrifuged smears showed the same cellularity in 54% of the cases and in 16% of the cases the cellularity in ACS was better than cytocentrifuged smears. In 30% of the cases cytocentrifuged smears had more cellularity as compared to ACS smears. Although the cellularity was less in these ACS smears, there was no problem in the interpretation of the smears. The yield of diagnostic cells was also same as that of cytocentrifuged smears.
The staining quality was optimal in 93% of ACS smears as compared to 92% of the cytocentrifuged smears. In 7% of the ACS smears and in 8% of cytocentrifuged smears the staining quality was suboptimal due to air drying.
All, 100% of the ACS smears were well-preserved and showed crisp and sharp cytomorphological details [Figure 2] whereas in 8% of the cytocentrifuged smears cellular distortion was noted resulting in bloating (false enlargement) of the nucleus.
|Figure 2: Adequate cellularity in urine specimen processed by ACS technique|
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In a few of the ACS smears (2%), especially in few fluid cases, the smears did show some greenish background staining; however, it did not obstruct the cytomorphological details of the diagnostic cells. The cytomorphological features were crisp and thus did not interfere in the interpretation of the smears [Figure 3]. This greenish background was due to the presence of the agar and it appeared similar to the presence of albumin that is used in the albumenized smears.
|Figure 3: Crisp cytomorphological details of malignant cells in serous effusion processed by ACS technique|
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For both the methods, the time needed for centrifugation is 10 min at 2000 rpm. The additional time for actual cytocentrifugation and processing by the new ACS technique is only 2 min, thus the total time taken by both ACS technique and the cytocentrifugation remains the same.
The additional cost of ACS smear is only 0.03 INR per smear as compared to the cytocentrifuged smear which is 12.5 INR in addition to the initial cost of 12.5 lakh INR for the machine.
| Discussion|| |
In cytology, to achieve our goal of good results the most important and basic step is the optimal smear preparation. In sparsely cellular samples especially the urine, CSF and sometimes serous effusions, one needs to use concentration techniques like cytocentrifugation or membrane filtration techniques to obtain a good representative smear. Cytocentrifugation is preferred method than the membrane filtration as it is easy and less tedious than membrane filtration.
In the absence of a cytocentrifuge machine, a smear has to be made directly from the centrifugate. However, these smears are often inadequate in cellularity, as the minimal number of cells present in the centrifugate gets spread on a larger area and are also lost during fixation of the smear for Papanicolaou staining.
In India, such sophisticated and expensive equipment like a cytocentrifuge is not always available in all laboratories, especially in the peripheral and rural set-ups. Thus, in these set-ups, it becomes difficult to achieve optimal representative smear in sparsely cellular smears.
Therefore, there is a need for a simple easily available cost-effective technique that can be used in the absence of cytocentrifuge for processing sparsely cellular samples.
Agar powder is easily available even in rural laboratories as it is widely used in microbiological studies. In this new ACS technique, a drop of 0.5% of agar solution is added to a drop of the centrifugate and smears are made from this suspension. About 0.5% agar does not solidify and remains jelly-like even at room temperature. It only changes the viscosity of the suspension, thereby trapping the cells, holding them together, which in turn becomes easy to make a smear, that has a maximum yield of the cells. Agar also prevents air drying of the smears resulting in optimal Papanicolaou staining.
The presence of agar in the ACS smears does not distort the cellular details and the cytomorphological features such as the nuclear chromatin, nuclear borders, nucleoli, cytoplasmic vacuoles, and cytoplasmic borders were crisp and sharp, thereby allowing proper interpretation of the smear. Although few Papanicolaou stained ACS smears showed greenish background staining it did not interfere with the interpretation as it was similar to that which is often seen in smears prepared on albumenized smears.
A larger study will further help in reemphasizing that the ACS method is useful in the absence of expensive equipment like cytocentrifuge and membrane filtration, especially in rural set-ups or at grassroots levels.
| Conclusion|| |
In conclusion, one can use the new ACS technique for preparing smears from sparsely cellular fluid samples in the absence of sophisticated machines, as it is simple, easy, yields good smears in terms of cellularity, cell preservation, the staining quality, and time required is also same.
Dr. (Mrs.) S. V. Kane.
Financial support and sponsorship
Cytopathology Department, Tata Memorial Hospital.
Conflicts of interest
There are no conflicts of interest.
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Swati B Dighe
Department of Cytopathology, Tata Memorial Hospital, Parel, Mumbai - 400012, Maharashtra
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]